Craig C. Malbon

Dynamic Complexes of β2-Adrenergic Receptors with Protein Kinases and Phosphatases and the Role of Gravin

Signals mediated by G-protein-linked receptors display agonist-induced attenuation and recovery involving both protein kinases and phosphatases. The role of protein kinases and phosphatases in agonist-induced attenuation and recovery of β-adrenergic receptors was explored by two complementary approaches, antisense RNA suppression and co-immunoprecipitation of target elements. Protein phosphatases 2A and 2B are associated with the unstimulated receptor, the latter displaying a transient decrease followed by a 2-fold increase in the levels of association at 30 min following challenge with agonist. Protein kinase A displays a robust, agonist-induced association with β-adrenergic receptors over the same period. Suppression of phosphatases 2A and 2B with antisense RNA or inhibition of their activity with calyculin A and FK506, respectively, blocks resensitization following agonist removal. Recycling of receptors to the plasma membrane following agonist-promoted sequestration is severely impaired by loss of either phosphatase 2B or protein kinase C. In addition, loss of protein kinase C diminishes association of phosphatase 2B with β-adrenergic receptors. Overlay assays performed with the RII subunit of protein kinase A and co-immunoprecipitations reveal proteins of the A kinase-anchoring proteins (AKAP) family, including AKAP250 also known as gravin, associated with the β-adrenergic receptor. Suppression of gravin expression disrupts recovery from agonist-induced desensitization, confirming the role of gravin in organization of G-protein-linked signaling complexes. The Ht31 peptide, which blocks AKAP protein-protein interactions, blocks association of β-adrenergic receptors with protein kinase A. These data are the first to reveal dynamic complexes of β-adrenergic receptors with protein kinases and phosphatases acting via an anchoring protein, gravin.

jun N-terminal Kinase Mediates Activation of Skeletal Muscle Glycogen Synthase by Insulin in Vivo

Mitogen-activated protein kinases (MAPKs) represent a conserved family of Ser/Thr protein kinases with central roles in intracellular signaling. Activation of three prominent members of the MAPK family, i.e. extracellular response kinases (ERK), jun N-terminal kinase (JNK), and p38, was defined in vivo in order to establish their role, if any, in the cardinal response of skeletal muscle to insulin, the activation of glycogen synthesis. Insulin was found to activate ERK, JNK, and p38 in skeletal muscle. The time courses for activation of the three MAPKs by insulin, however, are distinctly different. Activation of JNK occurs most rapidly, within seconds. Activation of p38 by insulin follows that of JNK, within minutes. Activation of ERK occurs last, 4 min after administration of insulin. The temporal relationship between the activation of ERK, JNK, p38 and the downstream elements p90rsk and PP-1 in vivo suggest that JNK, but neither ERK nor p38 MAPKs, mediates insulin activation of glycogen synthase in vivo. Activation of JNK by anisomycin in vivo mimics activation of glycogen synthase by insulin. Challenge by anisomycin and insulin, in combination, are not additive, suggesting a common mode of glycogen synthase activation. The p90rsk isoform rapidly activated by insulin is identified as RSK3. In addition, RSK3 can be activated by JNK in vitro. Based upon these data a signal linkage map for activation of glycogen synthase in vivo in skeletal muscle can be constructed in which JNK mediates activation of glycogen synthase via RSK3.

Phosphorylation of tyrosyl residues 350/354 of the beta-adrenergic receptor is obligatory for counterregulatory effects of insulin.

Insulin stimulates a loss of function and increased phosphotyrosine content of the β-adrenergic receptor in intact cells, raising the possibility that the β-receptor itself is a substrate for the insulin receptor tyrosine kinase. Phosphorylation of synthetic peptides corresponding to cytoplasmic domains of the β-adrenergic receptor by the insulin receptor in vitro and peptide mapping of the β-adrenergic receptor phosphorylated in vivo in cells stimulated by insulin reveal tyrosyl residues 350/354 and 364 in the cytoplasmic, C-terminal region of the β-adrenergic receptor as primary targets. Mutation of tyrosyl residues 350, 354 (double mutation) to phenylalanine abolishes the ability of insulin to counterregulate β-agonist stimulation of cyclic AMP accumulation. Phenylalanine substitution of tyrosyl reside 364, in contrast, abolishes β-adrenergic stimulation itself.

c-Jun Amino-terminal Kinase Is Regulated by Gα12/Gα13 and Obligate for Differentiation of P19 Embryonal Carcinoma Cells by Retinoic Acid

Retinoic acid induces P19 mouse embryonal carcinoma cells to differentiate to endoderm and increases expression of the heterotrimeric G-protein subunits Gα12 and Gα13. Retinoic acid was found to induce differentiation and sustained activation of c-Jun amino-terminal kinase, but not of ERK1,2 or of p38 mitogen-activated protein kinases. Much like retinoic acid, expression of constitutively active forms of Gα12and Gα13 induced differentiation and constitutive activation of c-Jun amino-terminal kinase. Expression of the dominant negative form of c-Jun amino-terminal kinase 1 blocked both the activation of c-Jun amino-terminal kinase and the induction of endodermal differentiation in the presence of retinoic acid. These data implicate c-Jun amino-terminal kinase as a downstream element of activation of Gα12 or Gα13 obligate for retinoic acid-induced differentiation.

Insulin-like Growth Factor Receptor-1 Stimulates Phosphorylation of the β2-Adrenergic Receptor in Vivo on Sites Distinct from Those Phosphorylated in Response to Insulin

G-protein-linked receptors have been shown to be substrates for growth factor receptors with intrinsic tyrosine kinase activity typified by the ability of insulin to both phosphorylate tyrosyl residues in the C terminus of and to counter-regulate the action of the β2-adrenergic receptor (Karoor, V., Baltensperger, K., Paul, H., Czech, M. P., and Malbon, C. C. (1995) J. Biol. Chem. 270, 25305-25308). Insulin-like growth factor-1 (IGF-1), another member of the growth factor family operating via receptors with intrinsic tyrosine kinase, is shown in the present work to stimulate in vivo the phosphorylation of the β2-adrenergic receptor. Analysis of tryptic digests prepared from phosphorylated β2-adrenergic receptors of IGF-1-treated, metabolically labeled smooth muscle cells was performed using reversed-phase high performance liquid chromatography, two-dimensional peptide mapping, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results of these separate analyses reveal that IGF-1 stimulates phosphorylation predominantly on tyrosyl residues Y132/141 of the second intracellular loop of the β2-adrenergic receptor rather than the C-terminal region targeted by the activated insulin receptor (Y350/354, Y364), although both growth factors block β-adrenergic agonist action. These data demonstrate selective phosphorylation of a G-protein-linked receptor by receptor tyrosine kinases for insulin and IGF-1 mapping to spatially distinct regions of this heptihelical membrane receptor.

Gsα Repression of Adipogenesis via Syk

Gsα regulates the differentiation of 3T3-L1 mouse embryonic fibroblasts to adipocytes, a process termed adipogenesis. Inducers of adipogenesis lead to a loss of Gsα and derepress differentiation to adipocytes. The broad spectrum tyrosine kinase inhibitor genistein is shown to block induction of adipogenesis, suggesting an early role of tyrosine phosphorylation in adipogenesis. Staining of phosphotyrosine identified prominent staining of a ∼70-kDa protein, hypothesized to be the tyrosine kinase Syk. Reverse transcription and polymerase chain reaction amplification established the expression of Syk mRNA in these embryonic fibroblasts. Immunoprecipitations with Syk-specific antibodies demonstrated the presence of Syk in fibroblasts and a rapid increase in the amount of phospho-Syk, peaking at 24 h post induction. Clones constitutively expressing Gsα, which can no longer be induced to differentiate, no longer display increased phospho-Syk levels in response to inducers. The linkage between Gsα and Syk was probed by immunoprecipitations revealing association of Syk with Gsα in the absence of induction. Upon induction of adipogenesis, Gsα levels decline and phospho-Syk levels as well as Syk kinase activity increase. Expression of wild-type Syk both potentiates the ability of inducers to act as well as induces adipogenesis itself. Expression of the kinase-deficient Syk had no such effects on adipogenesis. These data provide a new insight into the control of adipogenesis, suggesting that Gsα represses adipogenesis via Syk. Treatment with the inducers promotes a decline in Gsα, increases in levels of phospho-Syk, and adipogenesis.

Akt Mediates Sequestration of the β2-Adrenergic Receptor in Response to Insulin

The counterregulation of catecholamine action by insulin includes insulin-stimulated sequestration of the β2-adrenergic receptor. Herein we examined the signaling downstream of insulin receptor activation, focusing upon the role of 1-phosphatidylinositol 3-kinase and the serine-threonine protein kinase Akt (also known as protein kinase B) in the internalization of β2-adrenergic receptors. Inhibition of 1-phosphatidylinositol 3-kinase by LY294002 blocks insulin-induced sequestration of the β2-adrenergic receptor, implicating Akt in downstream signaling to the β2-adrenergic receptor. Phosphorylation studies of the C-terminal cytoplasmic domain of the β2-adrenergic receptor by Akt in vitroidentified Ser345 and Ser346 within a consensus motif for Akt phosphorylation. Double mutation (i.e.S345A/S346A) within this motif abolishes insulin counterregulation of β-adrenergic stimulation of cyclic AMP accumulation as well as insulin-stimulated sequestration. Furthermore, expression of constitutively activated Akt (T308D/S473D) mimics insulin action on cyclic AMP responses and β2-adrenergic receptor internalization. Expression of the dominant-negative version of Akt (K179A/T308A/S473A), in contrast, abolishes both insulin counterregulation of the cyclic AMP response as well as insulin-stimulated sequestration of the β2-adrenergic receptor. The action of the serine-threonine protein kinase Akt in insulin counterregulation mirrors the central role of protein kinase A in β-agonist-induced desensitization.

Conditional, Tissue-specific Expression of Q205L Gαi2 in Vivo Mimics Insulin Activation of c-Jun N-terminal Kinase and p38 Kinase

Deficiency of the G-protein subunit Gαi2 impairs insulin action (Moxham, C. M., and Malbon, C. C. (1996) Nature 379, 840–844). By using the promoter for the phosphoenolpyruvate carboxykinase gene, conditional, tissue-specific expression of the constitutively active mutant form (Q205L) of Gαi2 was achieved in mice harboring the transgene. Expression of Q205L Gαi2 was detected in skeletal muscle, liver, and adipose tissue of transgenic mice. Whereas the Gαi2-deficient mice displayed blunted insulin action, the Q205L Gαi2-expressing mice displayed enhanced insulin-like effects. Glycogen synthase in skeletal muscle was found to be activated in Q205L Gαi2-expressing mice, in the absence of the administration of insulin. Analysis of members of mitogen-activated protein kinase family revealed that both c-Jun N-terminal kinase and p38 are constitutively activated in vivo in the mice that express the Q205L Gαi2. ERK1,2, in contrast, are unaffected in the Q205L Gαi2-expressing mice. Insulin, like expression of Q205L Gαi2, activates both p38 and c-Jun N-terminal kinases as well as glycogen synthase. Activation of c-Jun N-terminal and p38 kinases in vivo with anisomycin, however, was insufficient to activate glycogen synthase. Much like Gαi2 deficiency provokes insulin resistance, expression of Q205L constitutively active Gαi2 mimics insulin action in vivo, sharing with insulin the activation of two mitogen-activated protein kinase members, p38 and c-Jun N-terminal kinases.

Insulin activation of mitogen-activated protein kinases Erk1,2 is amplified via beta-adrenergic receptor expression and requires the integrity of the Tyr350 of the receptor.

Insulin activates a complex set of intracellular responses, including the activation of mitogen-activated protein kinases Erk1,2. The counterregulatory actions of insulin on catecholamine action are well known and include phosphorylation of the β2-adrenergic receptor on Tyr350, Tyr354, and Tyr364 in the C-terminal cytoplasmic domain, as well as enhanced sequestration of the β2-adrenergic receptor. Both β-adrenergic agonists and insulin provoke sequestration of β2-adrenergic receptors in a synergistic manner. In the current work, cross-talk between insulin action and β2-adrenergic receptors revealed that insulin activation of Erk1,2 was amplified via β2-adrenergic receptors. In Chinese hamster ovary cells, expression of β2-adrenergic receptors enhanced 5–10-fold the activation of Erk1,2 by insulin and prolonged the activation, the greatest enhancement occurring at 5 min post-insulin. The potentiation of insulin signaling on Erk1,2 was proportional to the level of expression of β2-adrenergic receptor. The potentiation of insulin signaling requires the integrity of Tyr350 of the β2-adrenergic receptor, a residue phosphorylated in response to insulin. β2-adrenergic receptors with a Y350F mutation failed to potentiate insulin activation of Erk1,2. Expression of the C-terminal domain of the β2-adrenergic receptor (Pro323-Leu418) in cells expressing the intact β2-adrenergic receptor acts as a dominant negative, blocking the potentiation of insulin activation of Erk1,2 via the β2-adrenergic receptor. Blockade of β2-adrenergic receptor sequestration does not alter the ability of the β2-adrenergic receptor to potentiate insulin action on Erk1,2. We propose a new paradigm in which a G-protein-linked receptor, such as the β2-adrenergic receptor, itself acts as a receptor-based scaffold via its binding site for Src homology 2 domains, facilitating signaling of the mitogen-activated protein kinase pathway by insulin.

G-protein-linked receptors as tyrosine kinase substrates: new paradigms in signal integration.

Understanding how cells integrate signals from a variety of chemically diverse information-containing molecules into complex, orchestrated responses such as cell proliferation, differentiation and apoptosis is an overarching goal of cell biology. The ligand molecules that act upon cell surface receptors include those mediating proximal aspects of signal transduction through two major pathways: those that are G protein linked and those that are tyrosine kinase linked. G-protein receptors in the hundreds operate by means of less populous groups of heterotrimeric G proteins and the effectors regulated by G proteins. Growth factor receptors with intrinsic tyrosine kinase activity constitute a relatively large group of receptors, which share several downstream signalling elements with the G-protein-linked receptors. Integration between these two dominant pathways has been observed at several levels. The most proximal and intimate interaction possible--that between G-protein-linked receptors and tyrosine kinase receptors--has been discovered. Emerging data reveal new paradigms in which phosphorylation of G-protein-linked receptors on specific tyrosyl residues by tyrosine kinases enable G-protein-linked receptors to interact with adaptor molecules and enzymes previously thought to be restricted only to the signalling derivative of tyrosine kinase receptors.